Miller KE and Heald R (2015), Glutamylation of Nap1 modulates histone H1 dyna...

XB-ART-50580

J Cell Biol 2015 Apr 27;2092:211-20. doi: 10.1083/jcb.201412097.

Show Gene links Show Anatomy links

Glutamylation of Nap1 modulates histone H1 dynamics and chromosome condensation in Xenopus.

Miller KE , Heald R .

???displayArticle.abstract???
Linker histone H1 is required for mitotic chromosome architecture in Xenopus laevis egg extracts and, unlike core histones, exhibits rapid turnover on chromatin. Mechanisms regulating the recruitment, deposition, and dynamics of linker histones in mitosis are largely unknown. We found that the cytoplasmic histone chaperone nucleosome assembly protein 1 (Nap1) associates with the embryonic isoform of linker histone H1 (H1M) in egg extracts. Immunodepletion of Nap1 decreased H1M binding to mitotic chromosomes by nearly 50%, reduced H1M dynamics as measured by fluorescence recovery after photobleaching and caused chromosome decondensation similar to the effects of H1M depletion. Defects in H1M dynamics and chromosome condensation were rescued by adding back wild-type Nap1 but not a mutant lacking sites subject to posttranslational modification by glutamylation. Nap1 glutamylation increased the deposition of H1M on sperm nuclei and chromatin-coated beads, indicating that charge-shifting posttranslational modification of Nap1 contributes to H1M dynamics that are essential for higher order chromosome architecture.

???displayArticle.pubmedLink??? 25897082
???displayArticle.pmcLink??? PMC4411273
???displayArticle.link??? J Cell Biol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: h2ac21 mtor nap1l1 napsa tab3 ttll4
???displayArticle.antibodies??? H2a Ab2 Nap1l1 Ab1

???attribute.lit??? ???displayArticles.show???

	Figure 1. Nap1 binds linker histone H1M in Xenopus egg extracts and is required for normal chromosome condensation. (A) Coomassie-stained gel and Western blot of pull-down with recombinant Strep/His-tagged H1M protein (+) or Strep/His tag alone (−) from Xenopus egg extract. The two bands at 56 and 60 kD were recognized by a Nap1 antibody and identified by 1D liquid chromatography–tandem mass spectrometry as Nap1L1A and Nap1L1B (NCBI reference sequences NP_001082010 and NP_001080547). (B) Western blot of mock-depleted extract (dMock), Nap1-depleted extract (dNap1), and Nap1-depleted extract supplemented with 1 µM recombinant Nap1 (dNap1+ Nap1). β-Tubulin is shown as a loading control. (C) Fluorescence micrographs of spindles and mitotic chromosomes in mock-depleted extract, Nap1-depleted extract, and Nap1-depleted extract supplemented with 1 µM recombinant Nap1. (D) Individual mitotic chromosome dimensions in mock-depleted (dMock), Nap1-depleted (dNap1), H1M-depleted (dH1M) and Nap1-depleted extracts supplemented with 1 µM recombinant Nap1. Photographs on the left show representative images. Images were thresholded, and mean fiber lengths and widths were plotted. n ≥ 80 chromosomes from nine extracts in each condition; error bars are ± SEM, P < 0.001 from unpaired t test for length and breadth between dMock and dNap1, also between dNap1 and dNap1 + Nap1. SDs of chromosome lengths in each condition (µm): dMock = 7.37, dNap1 = 7.98, dNap1 + Nap1 = 6.52, and dH1 = 6.48. SDs of chromosome width in each condition (µm): dMock = 0.35, dNap1 = 0.31, dNap1 + Nap1 = 0.36, and dH1 = 0.23. The distribution of chromosome dimensions is also shown in histograms (Fig. S1, A and B). Bars, 10 µm.
	Figure 2. Nap1 is required for H1M-mediated chromosome condensation. (A) Representative Western blot of pelleted chromosomes (left) and cytoplasm (right) from mock- or Nap1-depleted egg extracts probed with antibodies to H1M and histone H2A. Levels of chromatin-bound H2A decreased by a mean of 0.8 ± 0.4% (mean ± SD) in Nap1-depleted extracts, whereas chromatin-bound H1M decreased by a mean of 48.6 ± 4.8%. Levels of H1M in Nap1-depleted cytoplasm decreased by 3.5 ± 1.2%, and levels of H2A decreased by 1.6 ± 0.2% on average. Quantification is shown in Fig. S1 C. (B) Immunofluorescence of mitotic chromosomes sedimented onto coverslips and stained for H1M. (C) Quantification of H1M/DNA fluorescence intensity on structures in B. Means ± SEM. Fluorescence intensity in each condition was background subtracted and normalized to H1-depleted extracts (see Materials and methods). n ≥ 50 structures were evaluated per condition in three separate extracts. *, P < 0.001 from unpaired t test comparing mock-depleted and Nap1-depleted extracts as well as comparing mock-depleted and Nap1-depleted/supplemented with Nap1N6C9. (D) Representative fluorescence micrographs of spindle assembly reactions in mock- or Nap1-depleted extracts supplemented with different concentrations of recombinant H1M. In the dNap1 + H1 condition, >99% of Hoechst-stained structures were hypercompacted chromatin masses with little or no microtubule polymerization around them. Bars, 10 µm.
	Figure 3. Glutamylation of Nap1 is required for mitotic chromosome condensation. (A) Schematic of the 393–amino acid Xenopus Nap1 protein, including three high-confidence glutamylation sites identified by 1D liquid chromatography–tandem mass spectrometry, marked with asterisks. Sites identified were N-terminal glutamate residues 30 and 31 as well as C-terminal residue 357. Three Nap1 mutants were created to reduce glutamylation by mutating indicated glutamic acid residues in the disordered N and C termini to aspartic acids. Nap1N6 = six glutamic acid residues mutated in the N terminus only. Nap1C9 = nine residues mutated in the C terminus only. Nap1 N6C9 = six N-terminal and nine C-terminal residues mutated together. Mutated sites are indicated in green. (B) Western blot of recombinant wild-type or mutant Nap1 proteins isolated from egg extracts and probed with antibodies that recognize polyglutamylated tubulin (anti-Glu). Nap1N6C9 displayed markedly reduced glutamylation in egg extracts with a 85 ± 3.8% decrease in band intensity (mean percentage ± SD, P = 0.005 from unpaired t test), whereas Nap1N6 and Nap1C9 were decreased by 19 ± 4.4% and 32 ± 5.1%, respectively. Recombinant wild-type Nap1 was pulled down in buffer as a negative control. (C) The Nap1 N6C9 mutant fails to rescue chromosome defects in Nap1-depleted egg extracts. Bar, 10 µm. (D) Chromosome length and width measurements were collected as in Fig. 2 A and averaged for each condition. n ≥ 60 chromosomes from five separate extracts. Error bars are ± SEM. P < 0.005 from unpaired t test comparing mock- and Nap1-depleted extracts as well as mock- and Nap1-depleted/supplemented with Nap1N6C9. SDs of chromosome lengths in each condition (µm): dMock = 6.0, dNap1 = 8.2, dNap1 + Nap1 = 6.9, and dNap1 + Nap1N6C9 = 6.9. SDs of chromosome width in each condition (µm): dMock = 0.34, dNap1 = 0.36, dNap1 + Nap1 = 0.34, and dNap1 + Nap1N6C9 = 0.38. The distribution of chromosome dimensions is also shown in histograms (Fig. S3, A and B).
	Figure 4. Glutamylation of Nap1 is required for proper H1 dynamics. (A) FRAP curves of H1M-GFP on mitotic chromosomes in metaphase-arrested egg extracts depleted of Nap1 (dNap1) and supplemented with either wild-type or the nonglutamylated mutant (Nap1N6C9). Curves represent means of n ≥ 10 chromosomes from three separate extracts. Half-lives for fluorescence recovery (t1/2 ± SD; s): dMock = 1.7 ± 0.68, dNap1 = 8.6 ± 0.51, dNap1+Nap1 = 1.7 ± 0.60, and dNap1±Nap1N6C9 = 9.5 ± 0.69. (B) FRAP curves of GFP-H1 on chromatin in metaphase- or interphase-arrested egg extracts depleted of Nap1 and supplemented with Nap1N6C9. Regardless of cell cycle state, Nap1-depleted extract supplemented with Nap1 N6C9 caused a similar significant reduction in H1M recovery time. Curves are means of n ≥ 10 chromosomes from five separate extracts. Half-times for fluorescence recovery (t1/2 ± SD; s): dMock-Mitosis = 1.9 ± 0.57, Nap1N6C9-Mitosis = 9.7 ± 0.67, dMock-Interphase = 2.1 ± 0.59, and Nap1N6C9-Interphase = 8.9 ± 0.70. For both A and B, the photobleach is plotted at time = 0.5 s. All curves were normalized to a baseline fluorescence of 1 at time = 0 s and 0 at time = 0.5 s. Error bars show ±SEM.
	Figure 5. Nap1 glutamylation promotes H1M deposition on sperm or on chromatin beads in extract and in buffer. (A) Representative Western blot of recombinant wild-type or mutant Nap1 proteins incubated with TTLL4 and probed with antibodies that recognize either Nap1 or glutamylated tubulin (anti-Glu). (B) Fluorescence micrographs of Xenopus sperm nuclei combined with 0.2 µM H1M-GFP alone or with equal concentrations of either Nap1 or Nap1N6C9, in the presence or absence of 1 µM TTLL4 enzyme. Bar, 10 µm. (C) Quantification of H1M/DNA fluorescence intensity on structures in B; means ± SD, n ≥ 50 structures were evaluated per condition in three separate experiments. , P < 0.001 in unpaired t test. (D) Western blot of isolated chromatin beads probed with antibodies against H1M. Left column shows chromatin beads assembled in H1M-depleted egg extracts showing that H1M is initially absent from the chromatin. H1M-depleted chromatin beads were then incubated in Nap1-depleted extracts supplemented with either recombinant Nap1 or the Nap1N6C9 mutant and 2 µM H1M-GFP. Greater amounts of both endogenous (E) and recombinant GFP-tagged H1M (R) were recruited to beads in the presence of wild-type Nap1 compared with the glutamylation site mutant. Histone H2A is shown as a loading control. (E) Western blot of isolated chromatin beads probed with antibodies against H1M in a similar experiment performed in buffer. H1M-depleted chromatin beads were incubated in Hepes buffer supplemented with either recombinant Nap1 or the Nap1N6C9 mutant and 2 µM H1M-GFP and the TTLL4 enzyme. A small amount of degraded H1M-GFP (D) is seen on the blot (arrow). Histone H2A is shown as a loading control. (F) Quantification of H1M-GFP band intensities in three experiments from three different extracts as in D; means ± SD. Band intensity for the (−)H1M condition was normalized to 0. , P = 0.005 by unpaired t test comparing wild-type and mutant conditions. (G) Quantification of mean H1M-GFP band intensities in three experiments as in E; means ± SD. *, P = 0.002 by unpaired t test comparing wild-type and mutant conditions.

References [+] :

Ausió, Histone variants--the structure behind the function. 2006, Pubmed